Cell division drama unfolds under the microscope
Separation of chromosomes during cell division in the Drosophila embryo. The DNA of the chromosomes is shown in red. During division, the chromosomes are attached to a cage-like structure at their midpoint, the 'centromere', which is shown here in green
It's the most dramatic step in cell division and the point of no return in this perfectly orchestrated series of events - the moment when the two sets of chromosomes are pulled apart as a single cell prepares to become two.
Now a collaboration between two groups of researchers in the Biochemistry Department has revealed details of how animal cells control entry into this part of the cell division cycle, known as anaphase.
The work provides a handle on the molecular events underlying cell division which are crucial to our understanding of how cancer develops.
Professors Kim Nasmyth and Ilan Davis and members of their groups have just published their findings in Nature Cell Biology1.
Researchers have known for some time that the two sets of newly replicated chromosomes are held together until they are ready to separate by a protein known as cohesin. Studies by Professor Nasmyth and others have demonstrated that in yeast, destroying cohesin is enough to trigger separation of the chromosomes.
Cohesin is also found in animal cells but researchers have puzzled over whether these cells might rely on a more complicated set of signals to trigger anaphase. The meagre amount of cohesin found on the chromosomes hints that this may be the case.
"Yeast has smaller chromosomes", says Dr Raquel Oliveira, the postdoctoral scientist in Professor Nasmyth's lab who carried out the work. 'So the question is, does this apply to higher organisms? The challenge to divide is very different from in a unicellular organism.'
To understand what is going on in these cells, Professor Nasmyth and his group looked for additional expertise to complement the tools they had available in their lab.
They wanted to be able to follow the chromosomes live under a microscope as the cells underwent division. So they joined forces with Professor Ilan Davis and his group in the department.
Nuclei dividing in an early stage Drosophila embryo. Each nucleus contains two sets of chromosomes which must be aligned neatly before they divide to opposite poles, forming two new daughter nuclei. DNA is shown in red and centromeres in green.
Professor Davis has developed very sensitive microscopy techniques to follow the live movement of molecules in Drosophila embryos. Using these techniques, a researcher can inject specific proteins into the embryo and look almost instantly at their real-time effects.
The early embryo lends itself perfectly to this type of work. It is actually a single cell, known as a syncytium. It forms when the fertilised egg undergoes rapid DNA duplication and division of the nucleus, where the chromosomes are contained, without separation of daughter cells.
To probe cohesin's role in the Drosophila embryo, Dr Oliveira used a technique which allows researchers to artificially destroy a selected protein. This had been developed in the lab by former graduate student Andrea Pauli.and requires two components, an enzyme called TEV and a short protein sequence that TEV recognises.
The sequence is first introduced into the target protein. When TEV comes along, it recognises the sequence and acts like molecular scissors by cutting at that specific spot.
Dr Oliveira used embryos from strains of Drosophila in which the cohesin protein had been altered so that it can be selectively destroyed by TEV. Before injecting the enzyme into the embryo though, she had to stop the natural progression of the cell into anaphase.
"We prevented the natural separation of the chromosomes and then cleaved cohesin at this stage", she explains. "We found that this triggered the physical separation of the chromosomes."
A Drosophila embryo blocked before anaphase and then injected with TEV to destroy cohesin. Soon after injection the chromosomes start to separate and are pulled to opposite poles. But the separation then goes awry and chromosomes begin jumping around the nucleus. Staining as for Movie 1.
But the way in which the chromosomes separated was far from normal. They behaved as if they had suddenly been released from a tight spring and were bounding around the nucleus.
"If you only cleave cohesin, the chromosomes start jumping from pole to pole in very fast movements. We wouldn't have spotted these defects without live cell imaging."
At this point, Dr Russell Hamilton, a bioinformatician in Professor Davis' lab, stepped in. He has developed a computer program which can be used to analyse these dynamic movements.
The program can take data from live imaging experiments and calculate the movement of molecules, such as chromosomes, in the cells. 'Particle Stats' takes the data from hundreds of molecules and calculates the average speed in just seconds.
Working closely together on the data, the researchers found that the chromosomes take much longer to separate than normal. The results pointed to some other signal being necessary for successful completion of anaphase.
Suspicion fell on the role of a protein called Cyclin dependent kinase 1 or Cdk1, known to be one of the key players in cell division.
Researchers know that during cell division, the cell must prevent Cdk1 from working if it is to go through anaphase properly. So Dr Oliveira and colleagues took an inhibitor of Cdk1 and injected this into the Drosophila embryo.
A Drosophila embryo blocked before anaphase and then injected with TEV and the Cdk1 inhibitor (called p27). When the two are combined, the chromosomes can separate as normal. Staining as for Movie 1.
"We did the combined blockage [of cohesin and Cdk1] and found that everything was normal. It was really amazing the way that we could reproduce what actually happens in the cell," says Dr Oliveira.
"We've been able to reproduce the full beauty of the movement and the kinetics of this complex morphological change - not just get some semblance of it - basically by artificial manipulation," adds Professor Nasmyth.
The experiments demonstrate that two key events, destruction of cohesin and inhibition of Cdk1, are all that is needed to drive the proper separation of chromosomes in anaphase.
Both researchers agree that the imaging system in Drosophila embryos, together with the availability of a program which can be used to look at chromosome movement, uniquely enabled them to address the question about what drives an animal cell through anaphase.
"When you want to interfere with protein function, the cleanest way is to inactivate exactly at the time when you want to know what the protein is doing", says Dr Oliveira. "It was great that Ilan was around and we could collaborate and do this together."
"Having a system where you can inject and observe the immediate consequences of what you've done is fantastic," Professor Nasmyth adds.
The groups are now looking at mammalian cells to see whether the same key events can drive these cells through the dramatic changes of anaphase.
- Oliveira, R. A., Hamilton, R. S., Pauli, A., Davis, I. and Nasmyth, K. Cohesin cleavage and Cdk inhibition trigger formation of daughter nuclei. Nature Cell Biology. Published online 17 January 2010, http://dx.doi.org/10.1038/ncb2018